1. Field of the Invention
The present invention is in the field of oligonucleotides. More particularly, the present invention is in the field of oligonucleotides which include the pyrazolo[3,4-d]pyrimidine analogs of the naturally occurring guanine and adenine bases. The oligonucleotides of the invention form triplexes with homopurine target runs in double stranded nucleic acids with increased effectiveness of binding when compared to triplex forming oligonucleotides which include the naturally occurring purine bases. The oligonucleotides of the invention can be used as sequence specific probes and in anti-gene therapy.
2. Brief Description of the Prior Art
Triplex forming oligonucleotides (ODNs) which bind to homopurine, or substantially homopurine sequences in double stranded DNA, are known in the prior art. To date three basic recognition motifs have been described for the sequence specific triplexation of ODNs to homopurine runs in double-stranded DNA. In each of the motifs the ODN resides in the major groove of the DNA duplex and hydrogen bonds to the bases of the homopurine strand. Because only two of the four DNA base pairs are recognized, the triplex forming ODNs are themselves usually composed of only two bases. The most frequently used recognition motif is based upon the formation of T-A-T and C-G-C triad wherein the third strand C residues are protonated (see Moser et al., Science 238: 645-650). In this motif (C.sup.+ /T or "pyrimidine motif") the triplex forming ODN has the same polarity as the homopurine strand of the target duplex. Because the cytosine residues on the triplex forming ODN must be protonated in order to hydrogen bond to guanine in the target, low pH facilitates the formation of these triplexes. Substituting 5-methyl cytidine (C*) for cytidine (C) can increase the stability of such triplexes at physiological pH (see Povsic et al. (1989) J. Amer. Chem. Soc. 111: 3059-3061).
A second recognition motif, first described for oligomeric triplexes by Hogan and co-workers, is based upon the formation of T-A-T and G-G-C triad. In this motif (G/T motif) the triplex forming ODN is antiparallel to the homopurine containing G-rich duplex strand (see Durland et al. Biochemistry 30: 9246-9255 (1991) ), and parallel to the homopurine duplex strand which is rich in A.
A third motif also has a third strand ODN which is antiparallel to the homopurine containing duplex stand. In this A/G motif (the "purine motif"), first described for oligomeric triplexes by Pilch et al. (1991) Biochemistry 30: 6081-6087, and Beal et al. (1991) Science 251: 1360-1363 A-A-T and G-G-C triads are formed. Triplexes based on both the G/T and A/G motifs are stable at physiologic pH.
U.S. Pat. No. 5,422,251 (Fresco) is directed to such triple stranded nucleic acids, where a homopurine or substantially homopurine run (containing approximately 10 purine bases) of a conventional Watson-Crick bonded nucleic acid duplex forms a hydrogen-bonded triplex with a third strand that has bases "corresponding" to bases of the homopurine run. The Fresco patent describes the rules of "correspondence" (which is differentiated from being "complementary" pursuant to Watson-Crick theory) in that it states that A of the homopurine run in the duplex can bind to A, U/T or I (inosine) in the third strand, and G of the homopurine run in the duplex can bind to I, G and C in the third strand. The Fresco patent also teaches that forming or utilizing triple stranded nucleic acids may be useful in diagnostic applications, control of gene expression and in control of genes in single cell and multi-cell organisms. The Fresco patent suggests that base residues which are "modified slightly (to form base analogues)" may also be incorporated in the triple-stranded nucleic acids of that patent.
Oligonucleotides which are complementary in the Watson Crick or triplex forming sense to a target sequence in single or double stranded DNA (as applicable) and also have an electrophilic cross-linking functionality designed to react with a nucleophilic site, (primarily the N-7 of guanine) have been described in publications and in international applications assigned to the same assignee as the present application (See WO 96/40711 and references cited therein, and Kutyavin et al. J. Amer. Chem. Soc. 115 9303-04 (1993)). These oligonucleotides exhibit sequence specific binding to the target sequence and can be used as sequence specific probes and in anti-gene therapy.
Another aspect of the pertinent background of the present invention relates to the antitumor antibiotic dextrorotatory (+) CC-1065, the structure of which is shown below. ##STR1##
As it can be seen, this antibiotic is composed of three repeating 1,2-dihydro-3H-pyrrolo[3,2-3-e]indole subunits. One of the subunits (CPI subunit) contains an electrophilic cyclopropyl moiety. The antibiotic is very stable in neutral aqueous solution. However, it binds strongly in the minor groove of A-T rich double stranded DNA and alkylates the DNA with resultant opening of the cyclopropyl ring. The following references describe or relate to the antibiotic CC-1065 and its CPI subunit:
Reynolds et al. (1986) J. Antibiotics (Tokyo), 39, 319-334; PA0 Boger et al. (1995) Proc. Natl. Acad. Sci. USA, 92, 3642-3649; PA0 Hurley et al. (1984) Science, 226, 843-844; PA0 Warpehoski et al., (1988) J. Med. Chem., 31, 590-603; PA0 Warpehoski et al. (1988) Chem. Res. Toxicol., 1, 315-333; PA0 Lin et al. (1991) Biochemistry, 30, 3597-3602; PA0 Reynolds et al. (1985) Biochemistry, 24, 6228-6237; PA0 Hurley et al. (1990) J. Am. Chem. Soc., 112, 4633-4649.
U.S. Pat. Nos. 4,400,518; 4,413,132; 4,423,229; 4,424,365; 4,496,492; 4,912,227; and 4,978,757 describe or relate to analogs of the antibiotic CC-1065, more particularly to analogs and derivatives of the CPI subunit of this antibiotic. These analogs are said to be useful primarily as bacteriostatic or bacteriocidal agents.
A significant disadvantage or problem encountered in connection with triplex forming oligonucleotides that are designed to bind to humopurine runs in the target by the GIT or A/G motif (and therefore are rich in guanine) is the tendency of guanine rich ODNs to self-associate. Self-association of the triplex forming ODN diminishes its effectiveness to bind to the the target double stranded DNA. Moreover, the nitrogen atom in the 7 position of the guanine base in nucleotides and oligonucleotides is a reasonably strong nucleophile. Therefore, triplex forming ODNs which also include a cross-linking agent, although definitely useful, may exhibit "self-alkylation" or "self-crosslinking" to a limited extent. Elimination of the "self-crosslinking" tendency would be considered an improvement, provided that sequence specific binding affinity to the target is maintained.
In light of the foregoing, it would be desirable to provide triplex forming ODNs which sequence specifically bind to target double stranded nucleic acids without the tendency of the guanine rich ODNs to self-associate, and when provided with a crosslinking functionality, self-crosslink in solution. This is because the lack of self-association and of self-crosslinking are likely to result in greater effectiveness of binding. Those skilled in the art will readily appreciate that as long as sequence specificity is retained, these qualities are advantageous for analytical, diagnostic, gene mapping, or like "probe" purposes, as well as for application in anti-gene therapy. The present application describes triplex forming ODNs which include the pyrazolo[3,4-d]pyrimidine analog of the natural guanine base and optionally the pyrazolo[3,4-d]pyrimidine analogs of adenine base as well, and provide the above-enumerated advantages